Temperature-responsive two-photon MOF-based fluorescent probe and its preparation method and application

Abstract

The present invention discloses a temperature-responsive two-photon MOF-based fluorescent probe, its preparation method, and application, relating to the field of fluorescent probe technology. MOF-199 is used as a nanocarrier, simultaneously loaded with a photothermal agent, gold nanorods (Au NRs), and a temperature-responsive pyridinium salt molecule (B). MOF-199 can degrade in the acidic tumor microenvironment, and the released copper ions react with overexpressed H2O2 through a Fenton-like reaction to generate ·OH, inducing tumor cell apoptosis and achieving chemodynamic therapy. Au NRs, with high photothermal conversion efficiency, can achieve photothermal therapy under near-infrared light irradiation, and local hyperthermia can further accelerate ·OH generation, thereby accelerating the chemodynamic therapy process. B, with its two-photon response characteristics, can achieve real-time temperature monitoring under near-infrared light irradiation, and the coated phase change material film can effectively prevent B leakage, achieving temperature-controlled release.

Description

Technical field:

[0001] The present invention relates to the technical field of fluorescent probes, and in particular to a temperature-responsive two-photon MOF-based fluorescent probe and a preparation method and application thereof. Background technology:

[0002] Cancer is a localized mass formed when normal cells, under the influence of various carcinogens, experience severe disruptions in the regulation of cell growth and proliferation. Current clinical treatments primarily rely on radiotherapy, chemotherapy, and surgical resection, which inevitably damage normal tissues, disrupt the immune system, and have a high recurrence rate. Therefore, the exploration of new cancer treatments is urgent.

[0003] With the advancement of science and technology, photothermal therapy (PTT) has attracted widespread attention as a novel phototherapy for tumors. Photothermal therapy utilizes light, radiation, microwaves, or other sources as heat sources to irradiate photothermal agents (PTAs). After absorbing the energy, the PTAs convert it into heat, causing a local increase in tissue temperature, thereby killing heat-sensitive cells and achieving therapeutic effects. Compared with traditional cancer treatments such as radiotherapy and chemotherapy, PTT offers the advantages of less invasiveness and greater specificity. However, during the process, excessively high or low temperatures may occur, causing thermal damage to surrounding normal tissue or failing to completely eliminate tumor cells. Therefore, determining the PTT temperature in situ to completely eliminate the tumor without causing thermal damage to surrounding normal tissue is a key issue in achieving the most effective PTT. Fluorescence thermometry is a noninvasive, fast-response, and highly sensitive method for measuring intracellular temperature. However, its further clinical application still faces challenges such as cytotoxicity, photobleaching, and interference from intracellular autofluorescence induced by ultraviolet light. Therefore, further research and improvement of fluorescence thermometry are needed to overcome these challenges and enable its clinical application.

[0004] In photothermal therapy, the applied laser needs to penetrate layers of tissue before reaching the target, so the interaction between light and tissue has a great impact on its efficacy. In the process of light passing through tissue, light reflection, light scattering and light absorption will occur. These interactions depend on the optical properties of the tissue and the wavelength of the light. Compared with ultraviolet light and visible light, the near-infrared (NIR) region is called the biological window. It has stronger penetration ability in tissues, less absorption / scattering, and deeper penetration depth. It is the most suitable light frequency for PTT. Therefore, designing a fluorescent temperature sensor with light-excited fluorescence characteristics in the near-infrared region is a favorable choice for achieving efficient PTT. Summary of the invention:

[0005] The technical problem to be solved by this invention is to provide a method for preparing a temperature-responsive two-photon MOF-based fluorescent probe. Using MOF-199 as a nanocarrier, it simultaneously loads gold nanorods (Au NRs), a photothermal agent, and temperature-responsive pyridinium salt molecules (B), enabling real-time temperature-guided noninvasive treatment. MOF-199 degrades in the acidic tumor microenvironment, releasing copper ions that react with overexpressed H2O2 via a Fenton-like reaction to generate ·OH, inducing tumor cell apoptosis and achieving chemodynamic therapy. Au NRs, with their high photothermal conversion efficiency, enable photothermal therapy under near-infrared light irradiation, and local hyperthermia further accelerates ·OH generation, thereby accelerating the chemodynamic therapy process. B, with its two-photon responsiveness, enables real-time temperature monitoring under near-infrared light irradiation, and a coated phase change material (protective agent) film effectively prevents B leakage, enabling temperature-controlled release.

[0006] The technical problem to be solved by the present invention is achieved by adopting the following technical solutions:

[0007] One of the purposes of the present invention is to provide a method for preparing a temperature-responsive two-photon MOF-based fluorescent probe, comprising the following steps:

[0008] (1) Preparation of pyridinium salt molecules:

[0009] a. reacting 4-methylpyridine, iodomethane and silver hexafluorophosphate to obtain an intermediate;

[0010] b. reacting the intermediate with 4-(N,N-diethyl)aminobenzaldehyde to obtain a pyridinium salt molecule;

[0011] (2) Preparation of Au NRs:

[0012] a. stirring and mixing tetrachloroauric acid, a reducing agent, and a surfactant dissolved in water to obtain a seed solution;

[0013] b. stirring and mixing tetrachloroauric acid, silver nitrate, a surfactant, and ascorbic acid dissolved in water to obtain a growth solution;

[0014] c. Add the seed solution to the growth solution and let it react to obtain Au NRs;

[0015] (3) Preparation of fluorescent probes:

[0016] MOF-199 was stirred and mixed with pyridinium salt molecules, and then Au NRs were added. The stirring was continued, and finally a protective agent was added to obtain a fluorescent probe.

[0017] Preferably, the molar ratio of 4-methylpyridine, methyl iodide and silver hexafluorophosphate is 1:(1-1.5):0.4.

[0018] Preferably, the molar ratio of the intermediate to 4-(N,N-diethyl)aminobenzaldehyde is 1:1.

[0019] Preferably, the molar ratio of tetrachloroauric acid (HAuCl4) to the reducing agent and surfactant is 0.01:2.4:400.

[0020] Preferably, the molar ratio of tetrachloroauric acid to silver nitrate, surfactant, and ascorbic acid is 0.01:0.096:200:1.87.

[0021] Preferably, the reducing agent is sodium borohydride (NaBH4).

[0022] Preferably, the surfactant is at least one of cetyltrimethylammonium bromide (CTAB), dodecyldimethylbenzylammonium chloride (DDBAC), and dodecyltrimethylammonium chloride (DTAC).

[0023] Preferably, the mass ratio of the MOF-199, pyridinium salt molecules, Au NRs, and protective agent is 1:1:(0.004-0.01):3.

[0024] Preferably, the protective agent is an organic phase change material. The function of the protective agent is to prevent the leakage of the ligand (pyridinium salt molecule) at room temperature. Further preferably, the organic phase change material is at least one of paraffin, fatty acid, fatty acid ester, and alcohol compound. Further preferably, the alcohol compound is at least one of dodecanol, tetradecanol, hexadecanol, octadecanol, and polyethylene glycol.

[0025] Preferably, the MOF-199 is synthesized from copper nitrate and benzene-1,3,5-tricarboxylate (BTC) in a molar ratio of 3:2.

[0026] A second object of the present invention is to provide a temperature-responsive two-photon MOF-based fluorescent probe prepared according to the preparation method.

[0027] The temperature-responsive two-photon MOF-based fluorescent probe of the present invention has a size of about 200 nm and can be accumulated at the tumor site through enhanced penetration and retention effects, laying a material foundation for tumor treatment.

[0028] The third object of the present invention is to provide the use of the temperature-responsive two-photon MOF-based fluorescent probe in the preparation of near-infrared excitation temperature-responsive tumor therapeutic drugs.

[0029] A fourth object of the present invention is to provide an application of the temperature-responsive two-photon MOF-based fluorescent probe in the preparation of a tumor photothermal-chemical combined therapy reagent.

[0030] The working principle of the temperature-responsive two-photon MOF-based fluorescent probe described in the present invention is as follows:

[0031] First, the temperature-responsive two-photon MOF-based fluorescent probe described in the present invention can convert light energy into heat energy under near-infrared laser irradiation to induce tumor cell death, and its fluorescence intensity has a good linear relationship with the system temperature under near-infrared laser irradiation, which can realize temperature monitoring during photothermal therapy.

[0032] Secondly, the temperature-responsive two-photon MOF-based fluorescent probe of the present invention dissociates into Cu in the acidic microenvironment of the tumor. 2+ It can mediate a Fenton-like reaction, decompose H2O2 to produce ·OH, and induce tumor cell death.

[0033] Furthermore, the increase in temperature during photothermal therapy can accelerate the Fenton-like reaction, thereby achieving synergistic effects between photothermal therapy and chemodynamic therapy and inducing tumor cell death more efficiently.

[0034] The beneficial effects of the present invention are as follows: the fluorescent probe preparation method provided by the present invention has the characteristics of readily available raw materials, low cost, and easy operation, and the prepared temperature-responsive two-photon MOF-based fluorescent probe has good biocompatibility and low dark toxicity, and can realize photothermal-chemical combined therapy, thereby more efficiently inducing tumor cell death. Description of the drawings:

[0035] Figure 1 Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) images of the temperature-responsive two-photon MOF-based fluorescent probe prepared in Example 1 of the present invention;

[0036] Figure 2 Two-photon excitation fluorescence spectra of the temperature-responsive two-photon MOF-based fluorescent probe prepared in Example 1 of the present invention at different excitation wavelengths;

[0037] Figure 3 (a) The two-photon excited fluorescence spectra of the temperature-responsive two-photon MOF-based fluorescent probe prepared in Example 1 of the present invention at different temperatures when excited by a 900 nm laser, and (b) the linear relationship between temperature and fluorescence intensity;

[0038] Figure 4 The temperature-responsive two-photon MOF-based fluorescent probe prepared in Example 1 of the present invention is shown in Figure 1 at different concentrations (a), different excitation powers (b), and a linear relationship diagram of the cooling stage -ln(θ) versus time (c);

[0039] Figure 5The present invention uses methylene blue to detect the UV-visible absorption spectrum of the temperature-responsive two-photon MOF-based fluorescent probe prepared in Example 1 to decompose H2O2 to produce ·OH (a); the electron spin resonance experiment is used to detect the ability of the temperature-responsive two-photon MOF-based fluorescent probe prepared in Example 1 to produce ·OH under different conditions (b);

[0040] Figure 6 Confocal microscopic images (a) of the temperature-responsive two-photon MOF-based fluorescent probe prepared in Example 1 of the present invention under 513 nm and 900 nm laser irradiation at different times (0 s, 30 s, 60 s) and the temperature calculated based on the linear relationship between fluorescence intensity and temperature change (b);

[0041] Figure 7 The MTT method was used to determine the cell viability under different conditions. Specific implementation method:

[0042] In order to make the technical means, creative features, objectives and effects achieved by the present invention easier to understand, the present invention is further described below with reference to specific embodiments and illustrations.

[0043] Example 1

[0044] Preparation of temperature-responsive two-photon MOF-based fluorescent probes:

[0045] 1. Synthesis of pyridinium salt molecules

[0046] a. Weigh 4-methylpyridine (9.3 g, 0.1 mol) and iodomethane (21.6 g, 0.15 mol) in toluene and stir at room temperature for 4 h. Then, reflux for 30 min, filter, and wash with ether to obtain a yellow solid. Add silver hexafluorophosphate (25.3 g, 0.1 mol) to the yellow solid, reflux for 30 min, filter, collect the filtrate, and concentrate to obtain an intermediate.

[0047] b. Measure 10 mL of anhydrous ethanol and add it to a 50 mL round-bottom flask. Weigh the intermediate (0.59 g, 5 mmol) and 4-(N,N-diethyl)aminobenzaldehyde (0.89 g, 5 mmol). Add a few drops of piperidine and reflux with stirring at 80°C for 10 h to obtain a red solution. Recrystallize it from ethanol and dry it in vacuum to obtain the pyridinium salt molecule.

[0048] 2. Preparation of MOF-199

[0049] Copper nitrate aqueous solution (0.9 mL, 0.1 M), hexadecyltrimethylammonium bromide aqueous solution (9.6 mL, 0.1 M), and BTC triethylamine salt aqueous solution (0.6 mL, 0.1 M) were added to a mixed solution of 15 mL of ethanol and 15 mL of deionized water, stirred vigorously for 10 min, centrifuged, washed with ethanol, and freeze-dried to obtain MOF-199.

[0050] 3. Preparation of Au NRs

[0051] a. Preparation of seed solution: Add HAuCl4 aqueous solution (0.1 mL, 2.5×10 -4 M) were mixed and stirred, and then an icy NaBH4 aqueous solution (0.6 mL, 0.01 M) was added, stirred vigorously for 2 min, and allowed to stand at room temperature for 2 h, and the solution turned brown.

[0052] b. Preparation of growth solution: Add AgNO3 aqueous solution (0.6 mL, 4 × 10 - 3 M) and HAuCl4 aqueous solution (1.0 mL, 2.5×10 -4 M), stirred at room temperature for 5 minutes, and then ascorbic acid aqueous solution (0.6 mL, 0.078 M) was added dropwise. The color of the mixed solution gradually turned colorless within 10 minutes. Then, 0.1 mL of seed solution was added to the mixed solution, and the mixture was allowed to stand at room temperature for 12 hours. The mixture was centrifuged and washed with deionized water. The product was collected and dispersed in 20 mL of deionized water to obtain an Au NRs dispersion.

[0053] 4. Preparation of temperature-responsive two-photon MOF-based fluorescent probes

[0054] 5 mg of MOF-199 and 5 mg of pyridinium salt molecules were weighed and added to 5 mL of deionized water, stirred at room temperature for 6 h, and then 5 mL of Au NRs dispersion was added and stirred for another 6 h. Then 15 mg of tetradecanol was added and stirred for another 3 h. The mixture was centrifuged, washed with methanol, and vacuum dried to obtain a temperature-responsive two-photon MOF-based fluorescent probe.

[0055] The morphology of the temperature-responsive two-photon MOF-based fluorescent probe prepared in Example 1 was characterized using scanning electron microscopy and transmission electron microscopy. Figure 1 As shown. Figure 1 It can be seen that the size of the temperature-responsive two-photon MOF-based fluorescent probe prepared in Example 1 is about 200 nm.

[0056] The temperature-responsive two-photon MOF-based fluorescent probe prepared in Example 1 was characterized by two-photon excitation fluorescence at different excitation wavelengths. Figure 2 As shown. Figure 2 It can be seen that the temperature-responsive two-photon MOF-based fluorescent probe prepared in Example 1 has excellent two-photon absorption performance.

[0057] The temperature-responsive two-photon MOF-based fluorescent probe prepared in Example 1 was subjected to two-photon excitation fluorescence characterization at different temperatures. Figure 3 As shown. Figure 3 It can be seen that the two-photon excited fluorescence intensity of the temperature-responsive two-photon MOF-based fluorescent probe prepared in Example 1 gradually decreases with increasing temperature, and has a good linear relationship.

[0058] Example 2

[0059] Detection of photothermal conversion efficiency in vitro:

[0060] Different concentrations (0, 50, 100 and 150 μg / mL) of the temperature-responsive two-photon MOF-based fluorescent probe prepared in Example 1 were added to 2 mL of ultrapure water and placed under 900 nm laser (1 W / cm 2 ) for 10 min. Then, the fixed concentration (150 μg / mL) was irradiated at different powers (0.8, 1 and 1.2 W / cm 2 ) laser irradiation for 10 minutes. At the same time, a near-infrared camera was used to record the temperature changes during this process. The results are shown in Figure 4 .

[0061] from Figure 4 It can be seen that under 900nm laser irradiation, when the probe concentration is 150μg / mL and the laser density is 1.2W / cm 2 After 10 minutes of illumination, the system temperature reached above 50°C, meeting the temperature requirement for photothermal therapy, laying the foundation for further in vivo photothermal therapy; and the photothermal conversion efficiency was calculated to be 58.3% through the linear data of the cooling stage -ln(θ) and time.

[0062] Example 3

[0063] By Cu 2+ Mediated Fenton-like reaction to generate ·OH

[0064] A solution containing 10 μg / mL methylene blue, 100 μM hydrogen peroxide, and 150 μg / mL of the temperature-responsive two-photon MOF-based fluorescent probe prepared in Example 1 was prepared. The degradation rate of MB induced by ·OH generated in the system was monitored by the absorbance change at 665 nm. The results are shown in FIG. Figure 5 .

[0065] from Figure 5It can be seen that the temperature-responsive two-photon MOF-based fluorescent probe prepared in Example 1 can achieve efficient Cu 2+ The mediated Fenton-like reaction produces ·OH, laying the foundation for the subsequent in vivo chemodynamic therapy.

[0066] Example 4

[0067] Intracellular temperature monitoring capabilities

[0068] Hep G2 cells incubated with the temperature-responsive two-photon MOF-based fluorescent probe prepared in Example 1 were irradiated with 513 nm and 900 nm lasers, respectively. Fluorescence results were obtained every 30 s. The intracellular temperature was monitored by the change in fluorescence intensity and the linear relationship between fluorescence intensity and temperature. The results are shown in Figure 6 .

[0069] from Figure 6 It can be seen that the temperature-responsive two-photon MOF-based fluorescent probe prepared in Example 1 can be used as a temperature probe to monitor the intracellular temperature in real time.

[0070] Example 5

[0071] Ability to induce cell death

[0072] The ability of the temperature-responsive two-photon MOF-based fluorescent probe to induce cell death was studied using the methylthiazolyl blue tetrazolium bromide (MTT) method. Before the experiment, Hep G2 cells were cultured in a 96-well plate. The cell culture medium was then mixed with different concentrations of the temperature-responsive two-photon MOF-based fluorescent probe (0, 50, 100, 150, 200, and 250 μg / mL) and incubated for 8 h. The probe was then illuminated with a laser (900 nm, 1 W / cm 2 ) irradiated for 15 min, and 100 μM H2O2 was added to the experimental group for co-incubation. Finally, the cell survival rate was calculated by measuring the absorbance at 490 nm. The results are shown in Figure 7 .

[0073] from Figure 7 It can be seen that under dark conditions, the cell survival rate is above 90%, indicating that the temperature-responsive two-photon MOF-based fluorescent probe prepared in Example 1 has low toxicity; under laser treatment, the cell survival rate is significantly reduced, indicating that the laser-induced photothermal process can effectively induce cell death. In addition, when H2O2 is added, the cell survival rate is further reduced, indicating that the dissociated Cu 2+ A Fenton-like reaction was carried out, producing highly toxic ·OH, which further synergistically induced cell death.

[0074] The basic principles, main features, and advantages of the present invention are shown and described above. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The above embodiments and descriptions are merely illustrative of the principles of the present invention. Various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention. Such changes and modifications are intended to fall within the scope of the present invention. The scope of protection claimed in the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing a temperature-responsive two-photon MOF-based fluorescent probe, characterized in that: The following steps are involved: (1) Preparation of pyridinium salt molecules: a. reacting 4-methylpyridine, iodomethane and silver hexafluorophosphate to obtain an intermediate; b. reacting the intermediate with 4-(N,N-diethyl)aminobenzaldehyde to obtain a pyridinium salt molecule; (2) Preparation of Au NRs: a. stirring and mixing tetrachloroauric acid, a reducing agent, and a surfactant dissolved in water to obtain a seed solution; b. stirring and mixing tetrachloroauric acid, silver nitrate, a surfactant, and ascorbic acid dissolved in water to obtain a growth solution; c. Add the seed solution to the growth solution and allow it to react to obtain Au NRs; (3) Preparation of fluorescent probes: MOF-199 and pyridinium salt molecules were stirred and mixed, and then Au NRs were added. The stirring was continued, and finally a protective agent was added to obtain a fluorescent probe. The protective agent is an organic phase change material; The organic phase change material is at least one of paraffin, fatty acid, fatty acid ester, and alcohol compound.

2. The preparation method according to claim 1, wherein: The molar ratio of the 4-methylpyridine, methyl iodide, and silver hexafluorophosphate is 1: (1-1.5): 0.4; the molar ratio of the intermediate to 4-(N,N-diethyl)aminobenzaldehyde is 1:

1.

3. The preparation method according to claim 1, wherein: The molar ratio of the tetrachloroauric acid to the reducing agent and the surfactant is 0.01:2.4:400; the molar ratio of the tetrachloroauric acid to the silver nitrate, the surfactant and the ascorbic acid is 0.01:0.096:200:1.

87.

4. The preparation method according to claim 1, wherein: The reducing agent is sodium borohydride; the surfactant is at least one of cetyltrimethylammonium bromide, dodecyldimethylbenzylammonium chloride, and dodecyltrimethylammonium chloride.

5. The preparation method according to claim 1, wherein: The mass ratio of the MOF-199, pyridinium salt molecules, Au NRs, and protective agent is 1:1:(0.004-0.01):

3.

6. The preparation method according to claim 1, wherein: The alcohol compound is at least one of dodecanol, tetradecanol, hexadecanol, octadecyl alcohol and polyethylene glycol.

7. The preparation method according to claim 1, wherein: The MOF-199 is synthesized from copper nitrate and benzene-1,3,5-tricarboxylate in a molar ratio of 3:

2.

8. A temperature-responsive two-photon MOF-based fluorescent probe prepared by the preparation method according to any one of claims 1 to 7.

9. Use of the temperature-responsive two-photon MOF-based fluorescent probe according to claim 8 in the preparation of near-infrared excitation temperature-responsive tumor therapeutic drugs.

10. Use of the temperature-responsive two-photon MOF-based fluorescent probe according to claim 8 in the preparation of a tumor photothermal-chemotherapy combined therapy agent.

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